Microgravity-induced bone deterioration is a major challenge in long-term spaceflights since the underlying mechanisms remain elusive. Previously, we reported that primary cilia of osteoblasts gradually disappeared in microgravity conditions, and cilia abrogation was necessary for the inhibition of osteogenesis induced by microgravity. However, the precise roles of primary cilia have not been fully elucidated. Here, we report that microgravity depolymerizes the microtubule network of rat calvarial osteoblasts (ROBs) reversibly but has no effect on the architecture of actin filaments. Preventing primary ciliogenesis by chloral hydrate or a small interfering RNA sequence (siRNA) targeting intraflagellar transport protein 88 (IFT88) effectively relieves microgravity-induced microtubule depolymerization, whereas the stabilization of microtubules using pharmacological approaches cannot prevent the disappearance of primary cilia in microgravity conditions. Furthermore, quantification of the number of microtubules emerging from the ciliary base body shows that microgravity significantly decreases the number of basal microtubules, which is dependent on the existence of primary cilia. Finally, microgravity-induced repression of the differentiation, maturation, and mineralization of ROBs is abrogated by the stabilization of cytoplasmic microtubules. Taken together, these data suggest that primary cilia-dependent depolymerization of microtubules is responsible for the inhibition of osteogenesis induced by microgravity. Our study provides a new perspective regarding the mechanism of microgravity-induced bone loss, supporting the previously established role of primary cilia as a sensor in bone metabolism.